Detection of chronic fatigue syndrome by decreased levels of RNase L inhibitor mRNA

ABSTRACT

Chronic fatigue syndrome in an individual is diagnosed by determining the level of RNase L inhibitor mRNA or protein in peripheral blood mononuclear cells. Significantly decreased levels of RLI mRNA or protein compared to healthy control individuals indicates the presence of chronic fatigue syndrome.

FIELD OF THE INVENTION

The present invention relates to the diagnosis of chronic fatiguesyndrome (CFS) by detecting decreased levels of mRNA. More specifically,the invention relates to the diagnosis of CFS by detection of RNase Linhibitor (RLI) mRNA. The RLI gene product is a component of theinterferon-mediated antiviral pathway.

BACKGROUND OF THE INVENTION

Chronic Fatigue Syndrome (CFS) is a systemic disorder defined by theCenters for Disease Control (CDC) as self-reported persistent orrelapsing fatigue lasting six or more months (Fukuda et al., Ann.Intern. Med., 121:953-959, 1994). Patients with CFS tend to haveindividualistic symptoms triggered by stress or unknown factorsincluding low grade fever, sore throat, headache, painful lymph nodes,muscle weakness, irritability, inability to concentrate, depression,irregular heartbeat and neuropsychological problems. Although theprecise nature and cause of CFS is unknown, there is some clinical andserological association with all of the human herpes viruses,particularly Epstein-Barr virus (EBV) and Human B-lymphotropic virus(HBLV). CFS may involve physiological manifestations of neurologicalinfluences on immune function by neurohormones or other immunomodulatorsof T-lymphocyte function. Upon binding to various lymphocyte surfaceantigens, viruses induce secretion of lymphokines which may interferewith immune response regulation including mucosal, humoral and cellularimmunity.

Many reports have described a decrease in natural killer (NK)cell-mediated cytotoxic activity and abnormal production of tumornecrosis factor- α (TNF-α), interleukin-1α (IL-1α) and interferon (IFN)(Klimas et al., J. Clin. Microbiol., 28:1403-1410, 1990; Morrison etal., Clin. Exp. Immunol., 83:441-446, 1991; Barker et al., Clin. Infect.Dis., 18:S136-S141,1994). The interferons area family of antiviral andantiproliferative cytokines which exert their pleiotropic effectsthrough the induction of several antiviral genes (Lengyel, Proc. Nati.Acad. Sci. USA, 90:5893-5895,1993; Pestka et al., Annu. Rev. Biochem.,56:727-777,1987). The 2-5-oligoadenylate (2-5A) system is anIFN-regulated, double stranded RNA (dsRNA) dependent pathway whichcontrols viral replication and cellular RNA stability. This pathway issummarized in FIG. 1. The primary enzyme in the 2-5A system is a 2-5oligoadenylate synthetase (2-5OAS) which converts ATP to 2'-5' linked2-5A oligomers (ppp(A2'p)_(n)), via a 2', 5' phosphodiester bond in thepresence of dsRNA (Ferbus et al., Biochem. Biophys. Res. Commun.,100:847-856, 1981; Mordechai et al., Virology, 206:913-922, 1995).Subnanomolar concentrations of 2-5A activate a latent endonuclease,RNase L, which is the terminal enzyme in the 2-5A system (Zhou et al.,Cell, 72:753-765, 1993). Activated RNase L degrades mRNA and rRNA on the3' side of a UpNp sequence, resulting in inhibition of viral andcellular protein synthesis (Floyd-Smith et al., Science, 212:1029-1032,1981; Wreschner et al., Nature, 289:414-417, 1981).

Elevated cellular levels of 2-5A and specific rRNA cleavage products arecorrelated with inhibition of encephalomyocarditis virus (EMCV)replication in IFN-treated cells, indicating that the antiviral andcytoprotective effect of IFN is partially mediated by the 2-5A system(Wreschner et al., ibid.; Silverman et al., Eur. J Biochem.,124:131-138, 1982). Introduction of 2-5A into cells inhibited cellulargrowth rates, implicating the involvement of the 2-5A system in theantiproliferative action of IFN. Increased concentrations of 2-5A andincreased RNase L activity have also been observed in individuals withCFS (Sudaholnik et al., In Vitro, 8:599-604, 1994).

RNase L is also regulated by a 68 kDa RNase L inhibitor (RLI) whichbinds to RNase L and inhibits the binding of 2-5A to RNase L (Bisbal etal., J. Biol. Chem., 270:13308-13317, 1995). RLI mRNA levels are notregulated by IFN, although IFN treatment of cells resulted in a threefold increase in RNase L and 2-5OAS mRNA levels.

There is currently no reliable diagnostic method for CFS. The difficultyassociated with establishing such a system and the consequent lack ofuniformity in patient samples studied, constitute major impediments inthe development of a treatment for CFS. The present invention providessuch a diagnostic method for CFS.

SUMMARY OF THE INVENTION

One embodiment of the present invention is a method for diagnosingchronic fatigue syndrome (CFS) in an individual, comprising:

isolating peripheral blood mononuclear cells (PBMC) from the individualand from a healthy control individual;

determining the amount of RNase L inhibitor mRNA present in the PBMCfrom the CFS individual and from the healthy individual; and

comparing the amount from the CFS individual to the amount from thecontrol individual, wherein a statistically significant decrease in theamount in the CFS patient compared to the control individual indicatesthe presence of CFS.

Preferably, the determining step comprises quantitative competitivepolymerase chain reaction or Northern blotting.

Another embodiment of the invention is a method for diagnosing chronicfatigue syndrome (CFS) in an individual, comprising:

isolating peripheral blood mononuclear cells (PBMC) from the individualand from a healthy control individual;

determining the amount of RNase L inhibitor protein in the PBMC from theCFS individual and from the healthy individual; and

comparing the amount from the CFS individual to the amount from thecontrol individual, wherein a statistically significant decrease in theamount in the CFS patient compared to the control individual indicatesthe presence of CFS.

Preferably, the determining step comprises Western blotting or ELISA.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram showing the 2-5oligoadenylate synthetasepathway. RLI=RNase L inhibitor; 2-5A=2', 5' oligoadenylates.

FIG. 2 is a graph comparing the level of RLI mRNA in patients having CFSwith control patients. RLI mRNA levels are expressed as copy number perμg total RNA. The horizontal bar indicates the mean for each group.

FIG. 3 is a graph comparing the level of RNase L mRNA in patients havingCFS as compared to control-individuals. RNase L mRNA levels areexpressed as copy number per μg total RNA. The horizontal bar indicatesthe mean for each group.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention includes the unexpected discovery that levels ofRLI mRNA and RLI protein are significantly down-regulated in lymphocytesof CFS patients compared to unaffected individuals. In the examplesdescribed below, RLI mRNA levels were determined by performingquantitative competitive PCR (Q/C PCR) on PCR-synthesized cDNA usingmRNA isolated from CFS or normal human lymphocytes. The Q/C PCR analysiswe conducted indicates a statistically significant decrease in RLI mRNApresent in the peripheral blood mononuclear cells (PBMC) of patientswith CFS as compared to RLI mRNA levels present in PBMC of healthyindividuals. Although the specific examples for the detection of RLImRNA levels were performed using Q/C PCR, one of ordinary skill in theart will appreciate that any other method capable of detecting RLI MRNAlevels is also within the scope of the present invention. One suchmethod is conventional Northern blotting, in which either total RNA ormRNA is isolated from PBMCs of an individual suspected of having CFS.The RNA is analyzed by agarose gel electrophoresis, transferred to anitrocellulose, nylon or other suitable membrane and incubated with alabeled probe complementary to a region of the RLI mRNA sequence.Because the RLI cDNA sequence is known, such probes can easily bedetermined and synthesized according to well known methods.

The levels of RLI protein present in the PBMCs of individuals suspectedof having CFS can also be determined using an immunoassay. Therefore, wehave used a polyclonal antibody generated against RLI in conjunctionwith conventional detection techniques such as Western blotting andenzyme-linked immunosorbent assay (ELISA). In particular, in Example 8,polyclonal antiserum specific for RLI has been produced which can beused to detect RLI protein levels by such methods.

Although differences in RLI mRNA and protein levels are observed in CFSversus healthy individuals, no such differences in RNase L levels areobserved. RLI mRNA deficiency in CFS patients may explain the increasein RNase L activity in CFS patients. The increased activation of RNase Lresults in a marked increase in cellular RNA turnover and constant 2',5' polymerization of ATP due to the virtually constitutive activation ofthe 2-5A pathway, resulting in general fatigue, myalgia and muscleweakness. RNase L, once activated, controls viral as well as cellularmRNA degradation. Unlike 2-5OAS, RNase L mRNA and protein levels do notfluctuate markedly during viral infection (Zhou et al., Cell,72:753-765; Salehzada et al., J. Biol. Chem., 268:7733-7740). RNase L isregulated post-transcriptionally by 2-5A molecules which act as anactivator (Floyd-Smith et al., Science, 212:1020-1032, 1981; Wreschneret al., Nature, 289:414-417, 1981) and by the formation of a latentheterodimeric protein--protein interaction with RLI (Bisbal et al., J.Biol. Chem. 270:13308-13317, 1995 and FIG. 1). In addition to bioactive2-5A, the control of RNase L activity in CFS individuals is most likelydue to the intracellular RNase L:RLI protein ratio.

The control of mRNA degradation is a critical element in the regulationof gene expression. The 2-5A system is a major pathway in controllingmRNA turnover via the effector enzyme RNase L. Upon viral infection oractivation of the IFN system, only the concentration of RNase L iselevated which may result is deregulation of the equilibrium betweenRNase L and RLI which promotes activation of the 2-5A system (FIG. 1).The data presented in the examples described below underscore thecentral role of the 2-5A system in the etiology of CFS.

The following examples provide illustrative methods for carrying outpreferred steps in conjunction with the practice of the presentinvention. As such, these examples are provided for illustrationpurposes only and are not intended to limit the invention.

PBMCs were isolated from both CFS and healthy individuals as describedin the following example.

EXAMPLE 1 Isolation of PBMCs

Twenty-five patients with CFS (15 males, 10 females) and 13 normalcontrol individuals (5 males, 8 females) were used as sources of PBMCs.All subjects met the epidemiological case definition of CFS establishedby the CDC. Controls were randomly selected volunteers employed byImmunosciences Lab, Inc., Beverly Hills, Calif., or had been seen byphysicians for routine physical examinations unrelated to the existenceof CFS or any other disorder. Venous blood (10 ml) was obtained byvenipuncture. PBMCs were isolated by Ficoll-Hypaque (Sigma, St. Louis,Mo.) gradient centrifugation of heparinized blood at 1,600 rpm for 30min. as described (Sudaholnik et al., Clin. Infect. Dis., 18:S96-S104,1994). The lymphocyte ring was isolated, rinsed twice withphosphate-buffered saline (PBS) and stained with Trypan Blue todetermine cellular viability.

RNA was isolated from PBMCs as described in the following example.

EXAMPLE 2 Isolation of RNA from PBMCs

RNA was extracted from PBMCs with TRIZOL™ reagent (GIBCO BRL,Gaithersburg, Md.) as described by the manufacturer. Briefly, PBMCs weresuspended in 1 ml TRIZOL™ reagent followed by addition of 200 μlchloroform. The cell suspension was shaken in an orbital shaker, thenincubated for 10 min at room temperature to allow phase separation.Samples were centrifuged (12,000 rpm, 10 min, 4° C.) and the aqueousphase was transferred to a fresh tube. Isopropanol (500 μl) was added toeach tube followed by incubation at -70° C. for 1 hour to precipitateRNA. Samples were centrifuged (12,000 rpm, 20 min., 4° C.) and theresulting RNA pellet was washed with 70% ethanol and dissolved in 10 μldiethyl pyrocarbonate (DEPC)-treated water. One μl was used forspectrophotometric quantitation. The RNA was treated with DNase I(amplification grade, GIBCO BRL) as described by the manufacturer.

Total RNA was converted to cDNA as described below.

EXAMPLE 3 Reverse transcription polymerase chain reaction (RT-PCR)

Total RNA was converted to cDNA using the GENE AMP™ RNA PCR kit(Perkin-Elmer, Norwalk, Conn.). Each reaction mixture (20 μl totalvolume) contained 10μl RNA (approx. 2 μg), 2.5 μM oligo d(T)₁₆, 1 mMdNTP, 5 mM MgCl₂, 2 μl 10×PCR buffer II, 1 μl MULV RNase inhibitor (10U/μl) and 2.5 μl MMLV reverse transcriptase (50 U/μl). Reaction mixtureswere incubated at 42° C. for 20 min, then ar 85° C. for 5 min.

Histone PCR was performed for each sample to serve as internal controlsfor intersample equivalency of total amounts of RNA analyzed asdescribed below.

EXAMPLE 4 Histone PCR

Aliquots (2 μl) of cDNA prepared according to Example 3 were mixed in atotal volume of 50 μl PCR reaction mixture containing 10×PCR buffer(United States Biochemical Corp., Cleveland, Ohio), 3 mM MgCl₂, 0.3 mMdNTP, 2.5 μl Taq DNA polymerase (5 U/μl) (U.S. Biochemical) and 1 μl (8pmol) of 5' and 3' histone amplified primer set5'-CCACTGAACTTCTGATTCGC-3'(SEQ ID NO: 1) and 5'-GCGTGCTAGCTGGATGTCTT-3'(SEQ ID NO: 2). The histone primers are complementary to the mRNA ofconstitutively expressed human histone gene H3.3 as described (Pieper etal., Cancer Commun., 2:13-20, 1990). The cDNA was amplified for 30cycles using the following parameters: 94° C. for 45 sec, 60° C. for 45sec, 72° C. for 90 sec in a PTC-100 Programmable DNA Thermal Cycler (MJResearch, Inc.). A sample with no added cDNA served as a negativecontrol.

Internal control DNA fragments were synthesized for use in Q/C PCR asdescribed in the following example.

EXAMPLE 5 Synthesis of internal RLI and RNase L control DNA fragments

Q/C PCR is an amplification technique based on a competitive approachusing non-homologous internal DNA standards which are DNA fragmentsconstructed for use in competitive PCR amplification for quantitation oftarget mRNA levels (Gilliland et al., Proc. Natl. Acad. Sci. U.S.A.,87:2725, 1990; Becker-Andre, Nucl. Acids Res., 17:9437, 1989; Siebert etal., BioTechniques, 11:244-249, 1993, the entire contents of which arehereby incorporated by reference). Each internal standard consists of aheterologous DNA fragment with primer templates that are recognized by apair of gene-specific (composite) primers. These templates "mimic" thetarget and are amplified during PCR. This mimic competes with the targetDNA for the same primers and thus acts as an internal standard.

To construct the internal standard, two rounds of PCR amplification areperformed. In the first PCR reaction, two composite primers are used,each of which contains the target gene primer sequence attached to ashort stretch of sequence designed to hybridize to opposite strands of a"mimic" DNA fragment. The desired primer sequences are thus incorporatedduring the PCR amplification. A dilution of the first PCR reaction isthen amplified again using only the gene-specific primers which ensuresthat all PCR mimic molecules have the complete gene-specific primersequences. The size of the PCR mimic can range from 200-650 base pairs,simply by choosing the appropriate sequences of the mimic DNA fragmentfor the composite primers. Following the second PCR amplification, thePCR mimic is purified by passage through a column.

Serial dilutions of PCR mimics are added to PCR amplification reactionscontaining constant amounts of the target cDNA sequence. The PCR mimicand target template thus compete for the same primers in the samereaction. By knowing the amount of PCR mimic added to the reactions, theamount of target template and initial mRNA levels can also bedetermined.

An internal control (mimic) DNA fragment (600 bp) was synthesized usingthe PCR MIMIC™ construction kit (Clontech, Palo Alto, Calif.) accordingto the manufacturer's instructions. Briefly, a 576 bp neutral fragment(BamHI/EcoRl fragment of the v-erbB gene) (SEQ ID NO: 3; 4 ng) was usedas a template with composite RLI or RNase L primers. RLI primers usedwere: 5'-GCCCCT-TTGGCGCCTTATCAATTGCGCMGTGAAATC-3' (SEQ ID NO: 4) and5'GTTTCGAGGGGTACCTGAGTTCGCGGATACCTCAACAGTGATACGG-3 (SEQ ID NO: 5). RNaseL primers used were:5'-GGACACGTAGAGGTCTTGAAGATTCCGCAAGTGAATAAATCTCCTCCG-3' (SEQ ID NO: 6)and 5'-CAATATGTCCCTCTACTTTCCAATACTGTCGCTCCGCCTTAATAC-3' (SEQ ID NO: 7).

PCR reaction mixtures (50 μl) were subjected to 20 cycles of PCR (94° C.for 45 sec, 60° C. for 45 sec, 72° C. for 90 sec) using the target RLIprimers in a final reaction volume of 100 μl. The PCR product waspurified using a pre-spun Chroma column (Clontech) as described by themanufacturer. The mimic DNA fragment was quantitated by electrophoresisand serial dilutions were prepared for Q/C PCR.

EXAMPLE 6 Quantitative Competitive PCR

Q/C PCR was performed essentially as described (Lion, Bone MarrowTransplantation, 14:505-509, 1994). Aliquots (0.2 μg) of cDNA were addedto serially diluted mimic DNA. The Q/C PCR reaction mixture (50 μl)contained the synthesized 600 bp RLI mimic DNA, 3 mM MgCl₂, 1.25 mMdNTP, 5 μl 10×PCR buffer (10 mM Tris-HCl, pH 8.3, 50 mM KCl, 20 mMMgCl₂), 2.5 units Taq DNA polymerase and 7 pmol (1 μl) of the RLIprimers (SEQ ID NOS: 4,5) or the RNase L primers (SEQ ID NOS: 6,7). TheQ/C PCR reaction mixtures were heated to 94° C. for 3 min and subjectedto 30 cycles of PCR (94° C. for 30 seconds, 54° C. for 30 seconds, 72°C. for 60 seconds). Following the final PCR cycle, a 5 min elongationstep was performed at 72° C. Aliquots (25 μl) of the PCR reaction wereanalyzed by electrophoresis on a 3% Nusieve/agarose (3:1)(FMC) gel inthe presence of 0.5 μl/ml ethidium bromide. The concentration of theinternal standard and RLI PCR products were measured by scanning theethidium bromide-stained gel using a digital imaging densitometer (AlphaInotech Corp.). Because the PCR-amplified RLI (712 bp) and RNase L (400bp) fragments are larger than their internal standard counterparts (600bp and 275 bp, respectively), they exhibit slower migration on anagarose gel. The concentration ratio of the internal controls divided bythe concentration of RLI were plotted against the input concentration ofthe internal standards per tube. The amount of RLI cDNA present in thereaction mixture was defined as the amount of internal control presentwhere the concentration ratio was equal to the PCR product.

RLI mRNA levels in PBMCs from 12 healthy controls ranged from 750 to5,000 RLI mRNA molecules per μg RNA (mean basal level=2296, S.E.=506).In contrast, the mean value for RLI mRNA present in PBMCs of CFSpatients (n=25) was significantly down-regulated compared to controlindividuals (CFS=569, S.E.=154) (P<0.0001, Mann-Whitney U-Test) (FIG.2). RNase L mRNA levels in control individuals ranged from 125-1,000mRNA molecules per μpg RNA (mean basal level=356, S.E.=63). The meanvalue for RNase L mRNA present in PBMCs of CFS patients (n=25) was notstatistically different compared to healthy control individuals(CFS=435, S.E.=67) (P<0.1, Mann-Whitney U-Test) (FIG. 3). Histone 3.3mRNA levels were tested in three normal and four CFS individuals andindicated that the same amount of RNA was analyzed in Q/C PCR.

These results demonstrate the down-regulation of RLI mRNA in CFSpatients as compared to healthy controls. CFS patients have increased2-5OAS activity which results in a marked increase in bioactive 2-5A (upto 220 fold compared to healthy controls) and RNase L activity(Sudaholnik et al., ibid.). The down-regulation of RLI mRNA in CFSindividuals may explain the increased RNase L activity observed in CFSpatients.

EXAMPLE 7 Detection of RLI mRNA by Northern blotting

Poly(A)⁺ RNA is isolated from both control and CFS RNA byoligo(dT)-cellulose chromatography. mRNA is analyzed by electrophoresison a 1.0% agarose-formaldehyde gel. The mRNA is transferred to anitrocellulose membrane using a conventional Northern blottingapparatus, followed by prehybridization and hybridization with a ³²P-labeled RLI cDNA probe. The filter is then washed under highstringency conditions and exposed to x-ray film. The resulting bands arequantitated using a scanning densitometer. The results show that RLImRNA levels are significantly decreased in CFS individuals compared tohealthy control individuals.

As described above, CFS can also be diagnosed by evaluating levels ofRLI protein. One method of determining these levels involves the use ofan immunoassay for the protein. Both polyclonal and monoclonalantibodies can be used; however, polyclonal antibodies are preferred soas to account for antigenic variation among individuals. We preparedpolyclonal antibodies as described in the following example.

EXAMPLE 8 Generation of polyclonal antiserum against RLI

The peptide DKCKPKKCRQECKKS (SEQ ID NO: 8), corresponding to amino acids14-28 of RLI (Bisbal et al., ibid.), was synthesized on an automatedpeptide synthesizer using methods well known to one of ordinary skill inthe art. The peptide was then conjugated to the immunogenic carrierprotein keyhole limpet hemocyanin (KLH) with glutaraldehyde. Thepeptide-KLH conjugate was injected into three Flemish Giants rabbits.Each animal received six subcutaneous injections (100 μg/injection) atsix distinct sites. Booster injections were given at 2, 4, 6, 8, 10 and12 weeks. The rabbits were bled at 10, 11, 12 and 14 weeks and the blood(60 ml/bleed) was centrifuged to pellet blood cells. The remaining serum(25-30 ml) was tested for immunoreactivity against RLI on a Western blotof lymphocyte lysate from CFS patients, as described in the example thatfollows.

EXAMPLE 9 Detection of RLI by Western blotting

PBMCs from CFS patients and control individuals are lysed in buffercontaining 20 mM HEPES, pH 7.5, 5 mM MgCl₂, 120 mM KCI, 10% glycerol,0.5% Nonidet-P40™ (NP-40) as described (Mordechai et al., Virology,206:913-922, 1995). Proteins are separated by SDS-polyacrylamide gelelectrophoresis (SDS-PAGE). The separated proteins areelectrophoretically transferred to nitrocellulose. The nitrocellulose isthen incubated with the polyclonal antibody to RLI produced as describedin Example 8. The primary antibody is replaced with anti IgG-horseradishperoxidase conjugate (Amersham). Bound secondary antibody-horseradishperoxidase conjugate is detected using chemiluminescence detectionreagents (Amersham). There is significantly more RLI present in PBMCsfrom control individuals compared to CFS individuals.

Any of a variety of other immunoassays can also be adapted to determineRLI protein levels. The following example provides one such assay in theform of an ELISA.

EXAMPLE 10 Detection of RLI by ELISA

Serial dilutions of clarified cell lysate from Example 8 are placed in96-well microtiter plates and incubated for 3 hours at 37° C. Lysate isremoved, and the wells are washed three times with PBS. Polyclonalantibody against RLI is added to the wells and incubated for 1 hour atroom temperature. Antibody is removed and the wells are washed severaltimes with PBS. Alkaline phosphatase-conjugated goat anti-rabbit IgG isthen added to the wells and incubated for 30 min at room temperature,followed by addition of a colorimetric alkaline phosphatase substrate.The plates are read at on a microplate reader set at the optimumwavelength of the colorimetric reagent. PBMC lysates from CFSindividuals contain significantly more RLI than do normal lysates.

It should be noted that the present invention is not limited to onlythose embodiments described in the Detailed Description. Any embodimentwhich retains the spirit of the present invention should be consideredto be within its scope. However, the invention is only limited by thescope of the following claims.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 8                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       CCACTGAACTTCTGATTCGC20                                                        (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       GCGTGCTAGCTGGATGTCTT20                                                        (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 576 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       GGATCCCCGCAAGTGAAATCTCCTCCGTCTTGGAGAAGGGAGAGCGTTTGCCCCAGCCAC60                CCATTTGTACCATTGATGTGTACATGATCATGGTCAAATGCTGGATGATTGATGCAGACA120               GCCGTCCCAAGTTTCGTGAGCTGATTGCAGAGTTCTCCAAAATGGCTCGTGACCCTCCCC180               GCTATCTTGTTATACAGGGAGATGAAAGGATGCACTTGCCTAGCCCTACAGATTCCAAGT240               TTTATCGCACCCTGATGGAGGAGGAGGACATGGAAGACATTGTGGATGCAGATGAGTATC300               TTGTCCCACACCAGGGCTTTTTCAACATGCCCTCTACATCTCGGACTCCTCTTCTGAGTT360               CATTGAGCGCTACTAGCAACAATTCTGCTACAAACTGCATTGACAGAAATGGGCAGGGGC420               ACCCTGTGAGGGAAGAGGCTTCCTGCCTGCTCCAGAGTATGTAAACCAGCTGATGCCCAA480               GAAACCATCTACTGCCATGGTCCAGAATCAAATCTACAACTTCATCTCTCTCACAGCAAT540               CTCAAAGCTCCCCATGGACTCAAGATACCAGAATTC576                                       (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 37 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       GCCCCTTTGGCGCCTTATCAATTGCGCAAGTGAAATC37                                       (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 46 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       GTTTCGAGGGGTACCTGAGTTCGCGGATACCTCAACAGTGATACGG46                              (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 48 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       GGACACGTAGAGGTCTTGAAGATTCCGCAAGTGAATAAATCTCCTCCG48                            (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 45 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       CAATATGTCCCTCTACTTTCCAATACTGTCGCTCCGCCTTAATAC45                               (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       AspLysCysLysProLysLysCysArgGlnGluCysLysLysSer                                 151015                                                                        __________________________________________________________________________

What is claimed is:
 1. A method for detecting an increased likelihood ofthe presence of chronic fatigue syndrome (CFS) in an individual,comprising:isolating peripheral blood mononuclear cells (PBMCs) fromsaid individual; and determining the amount of RNase L inhibitor (RLI)mRNA present in said PBMCs from said individual, wherein the presence ofan amount of RLI MRNA less than about 750copies/μg RNA indicates anincreased likelihood of the presence of CFS.
 2. The method of claim 1,wherein said determining step comprises quantitative competitivepolymerase chain reaction.
 3. The method of claim 1, wherein saiddetermining step comprises Northern blotting.
 4. The method of claim 1,wherein the presence of an amount of RLI mRNA less than about 700copies/μg RNA indicates an increased likelihood of the presence of CFS.